百脉根离子通道蛋白POLLUX及其与翻译延伸因子EF1A相互作用的研究
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摘要
根瘤菌与豆科植物相互作用早期信号转导成为近年来共生体系研究的热点问题。共生信号转导研究中,苜蓿和百脉根两种模式植物部分关键信号转导基因相继克隆。对这些关键基因功能和作用机制研究,揭示其相互调控因素,有利于阐明共生信号转导的分子机制。酵母双杂交技术为搜寻信号转导基因调控因子提供了一条有利途径。
离子通道蛋白POLLUX是共生信号途径上游的关键功能基因,其突变后,百脉根除不能结瘤外,生理上失去了钙离子振荡表型。本实验以以百脉根POLLUX膜内区段(C-Pollux)为模板,设计引物构建了诱饵质粒pGBKT7-C-Pollux。以pGBKT7-C-Pollux为诱饵,筛选百脉根酵母双杂交文库,获得翻译延伸因子EF1A等多个与POLLUX膜内区段相互作用的克隆子。酵母回转实验、测序及NCBI-BLAST分析,表明翻译延伸因子EF1A与离子通道蛋白POLLUX膜内区段(C-POLLUX)相互作用。
依据百脉根数据库BLAST分析得到的序列,设计cDNA引物,RT-PCR扩增得到翻译延伸因子EF1A全长cDNA。表达翻译延伸因子EF1A及其两个截短区段ED219、ED267和离子通道蛋白C-POLLUX,并利用相应琼脂糖凝胶珠分离纯化目的蛋白。通过Pull-down和Western Blotting实验证明离子通道蛋白C-POLLUC与翻译延伸因子EF1A存在相互作用。利用小样rnating方法,检验其他关键共生信号转导基因与翻译延伸因子在酵母体内相互作用,表明结瘤因子受体NFR1,共生受体激酶SYMRK和离子通道蛋白CASTOR与EF1A在酵母体内存在相互作用,而结瘤因子受体NFR5、钙和钙调素结合蛋白CCaMK与翻译延伸因子EF1A在酵母体内不能够相互作用。
The early signaling transduction of symbiosis system in which the Rhizobium interacting legume plants with each others has been most focused on by global researchers. Multi of signaling key genes has been cloned from the two model plants, Medicago truncatula and Lotus japonicus. To confirm the function and action mechanism, and how to inter-regulate of these genes, are useful for understanding the molecular mechanism of the symbiosis system. It's a useful tool utilizing Yeast two-hybrid assay to find the regulation factor for the signaling genes.
Cation channel protein, POLLUX had function as an important role in the upstream symbiotic signaling net. The pollux mutant, could not form nodules and was lack of calcium spiking phenotype, physically. We constructed BD-C-Pollux bait plasmid, and used it to screen the Lotus Yeast two-hybrid cDNA library. There were lots of clones including the translation elongation factor 1A acquired from library interacting with POLLUX. The positive clones were identified by mating in yeast again, then sequenced and searched by program BLAST on NCBI net. We confirmed that the translation elongation factor 1A (EF1A) interacted with C-POLLUX.
Then we designed the primers of EF1A according to the sequence on the database of Lotus japonicus, isolated the total RNA, obtained of the translation elongation factor 1A full length cDNA by RT-PCR. To express the protein in E.coli, the fusion tag plasmid were constructed, such as pET28a-EF1A, pET28a-ED219, pET28a-ED267, pGEX-6p1-C-Pollux, and then the fusion proteins were purified with the suitable beads. Pull down and Western blotting assays confirmed the interaction between C-POLLUX and EF1A in vitro. And further more EF1A interacting with other signaling genes were checked by small scale mating in yeast cell, the results were including that NFR1, SYMRK and CASTOR interacted with EF1A in yeast cells while NFR5 or CCaMK did not.
离子通道蛋白POLLUX是共生信号途径上游的关键功能基因,其突变后,百脉根除不能结瘤外,生理上失去了钙离子振荡表型。本实验以以百脉根POLLUX膜内区段(C-Pollux)为模板,设计引物构建了诱饵质粒pGBKT7-C-Pollux。以pGBKT7-C-Pollux为诱饵,筛选百脉根酵母双杂交文库,获得翻译延伸因子EF1A等多个与POLLUX膜内区段相互作用的克隆子。酵母回转实验、测序及NCBI-BLAST分析,表明翻译延伸因子EF1A与离子通道蛋白POLLUX膜内区段(C-POLLUX)相互作用。
依据百脉根数据库BLAST分析得到的序列,设计cDNA引物,RT-PCR扩增得到翻译延伸因子EF1A全长cDNA。表达翻译延伸因子EF1A及其两个截短区段ED219、ED267和离子通道蛋白C-POLLUX,并利用相应琼脂糖凝胶珠分离纯化目的蛋白。通过Pull-down和Western Blotting实验证明离子通道蛋白C-POLLUC与翻译延伸因子EF1A存在相互作用。利用小样rnating方法,检验其他关键共生信号转导基因与翻译延伸因子在酵母体内相互作用,表明结瘤因子受体NFR1,共生受体激酶SYMRK和离子通道蛋白CASTOR与EF1A在酵母体内存在相互作用,而结瘤因子受体NFR5、钙和钙调素结合蛋白CCaMK与翻译延伸因子EF1A在酵母体内不能够相互作用。
The early signaling transduction of symbiosis system in which the Rhizobium interacting legume plants with each others has been most focused on by global researchers. Multi of signaling key genes has been cloned from the two model plants, Medicago truncatula and Lotus japonicus. To confirm the function and action mechanism, and how to inter-regulate of these genes, are useful for understanding the molecular mechanism of the symbiosis system. It's a useful tool utilizing Yeast two-hybrid assay to find the regulation factor for the signaling genes.
Cation channel protein, POLLUX had function as an important role in the upstream symbiotic signaling net. The pollux mutant, could not form nodules and was lack of calcium spiking phenotype, physically. We constructed BD-C-Pollux bait plasmid, and used it to screen the Lotus Yeast two-hybrid cDNA library. There were lots of clones including the translation elongation factor 1A acquired from library interacting with POLLUX. The positive clones were identified by mating in yeast again, then sequenced and searched by program BLAST on NCBI net. We confirmed that the translation elongation factor 1A (EF1A) interacted with C-POLLUX.
Then we designed the primers of EF1A according to the sequence on the database of Lotus japonicus, isolated the total RNA, obtained of the translation elongation factor 1A full length cDNA by RT-PCR. To express the protein in E.coli, the fusion tag plasmid were constructed, such as pET28a-EF1A, pET28a-ED219, pET28a-ED267, pGEX-6p1-C-Pollux, and then the fusion proteins were purified with the suitable beads. Pull down and Western blotting assays confirmed the interaction between C-POLLUX and EF1A in vitro. And further more EF1A interacting with other signaling genes were checked by small scale mating in yeast cell, the results were including that NFR1, SYMRK and CASTOR interacted with EF1A in yeast cells while NFR5 or CCaMK did not.
引文
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2.尚忠林,毛国红,孙大业.植物细胞内钙信号的特异性.植物生理学通讯,2003,39(2)：93-99
3.沈世华,荆玉祥.中国生物固氮研究现状和展望.科学通报,2003,48(6)：535-540
4.肖梅,张洪义,周宁新.离子通道病的研究进展.空军总医院学报,2005,21(4)：219-221
5.杨绮.百脉根共生信号途径中CASTOR及其相互作用蛋白的研究[D]；华中农业大学；2007
6.曾昭海,胡跃高.共生固氮在农牧业上的作用及影响因素研究进展.中国生态农业学报,2006,14(4)：21-24
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26. Lerouge P, Roche P, Faucher C, et al. Symbiotic host-specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal. Nature,1990,344:781-784
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2.尚忠林,毛国红,孙大业.植物细胞内钙信号的特异性.植物生理学通讯,2003,39(2)：93-99
3.沈世华,荆玉祥.中国生物固氮研究现状和展望.科学通报,2003,48(6)：535-540
4.肖梅,张洪义,周宁新.离子通道病的研究进展.空军总医院学报,2005,21(4)：219-221
5.杨绮.百脉根共生信号途径中CASTOR及其相互作用蛋白的研究[D]；华中农业大学；2007
6.曾昭海,胡跃高.共生固氮在农牧业上的作用及影响因素研究进展.中国生态农业学报,2006,14(4)：21-24
7.周冰,曹诚,刘传暄.翻译延伸因子1A的研究进展.生物技术通讯.2007,281-284
8. Amor B B, Shaw S L, Oldroyd G E, Maillet F, Penmetsa R V, Cook D, Long S R, Denarie J, Gough C. The NFP locus of Medicago truncatura controls an early step of Nod factor signal transduction upstream of a rapid calcium flux and root hair deformation. Plant J,2003,34:495-506
9. Ane J M, Kiss G B, Riely B K, Penmetsa R V, Oldroyd G E, Ayax C, Levy J, Debelle F, Baek J M, Kalo P et al. Medicago truncatula DMI1 required for bacterial and fungal symbioses in legumes. Science,2004,303:1364-1367
10. Broughton W J, Jabbouri S, Perret X. Keys to symbiotic harmony. Journal of Bacteriology,2000,182: 5641-5652
11. Capoen W, Goormachtig S, De Rycke R, Schroeyers K, Holsters M, SrSymR K. a plant receptor essential for symbiosome formation. Proc Natl Acad Sci USA,2005,102:10369-10374
12. Carroll B J, McNeil D L, Gresshoff P M. A Supernodulation and Nitrate-Tolerant Symbiotic (nts) Soybean Mutant. Plant Physiol,1985,78(1):34-40
13. D'Haeze W, Holsters M. Nod factor structures, responses, and perception during initiation of nodule development. Glycobiology,2002,12:79-105
14. Denztie J, Cullimore J. Lipooligosaccharide nodulation factors:A minnireview new class of signaling molecules mediating recognition and morphogenesis. Cell,1993,74:951-954
15. Denarie J. Rhizobium lipo-chitooligosaccharide nodulation factor:Signaling molecules mediating recognition and morphogenesis. Annu Rev Biochem,1996,65:503-535
16. Ehrhardt D W, Wais R,Long S R. Calcium spiking in plant root hairs responding to Rhizobium nodulation signals. Cell,1996,85:673-681
17. Endre G, Kereszt A, Kevei Z, Mihacea S, Kalo P, Kiss G B. A receptor kinase gene regulating symbiotic nodule development. Nature,2002,417:962-966
18. Heckmann A B, Lombardo F, Miwa H, Perry J A, Bunnewell S, Parniske M, Wang T L, Downie J A. Lotus japonicus Nodulation Requires Two GRAS Domain Regulators, One of Which Is Functionally Conserved in a Non-Legume. Plant Physiology,2006,142:1739-1750
19. Heidstra R, Bisseling T. Nod factor-induced host resoponses and mechanisms of Nod factor perception. New Phytol,1996,133:25-43
20. Hubert H Felle, Eva Kondorosi, Adim Kondorosi, et al. Nod factors modulate the concentration of cytosolid free calcium differently in growing and non-growing root hairs of Medicago Sativa L. Planta,1999,209:207-212
21. Imaizumi-Anraku H, Takeda N, Charpentier M, Perry J, Miwa H, Umehara Y, Kouchi H, Murakami Y, Mulder L, Vickers K, Pike J, Downie J. A, Wang T, Sato S, Asamizu E, Tabata S, Yoshikawa M, Murooka Y, Wu G J, Kawaguchi M, Kawasaki S, Parniske M and Hayashi M. Plastid proteins crucial for symbiotic fungal and bacterial entry into plant roots. nature,2005,433 (7025):527-31
22. Izawa T, Fukata Y, Kimura T, et al. Elongation factor-1-alpha is a novel substrate of rho-associated kinase[J]. Biochem Biophys Res Commun,2000,278(1):72
23. Kalo P, Gleason C, Edwards A, Marsh J, Mitra R M, Hirsch S, Jakab J, Sims S, Long S R, Rogers J et al. Nodulation signaling in legumes requires NSP2, a member of the GRAS family of transcriptional regulators. Science,2005,308:1786-1789
24. Kanamori N, Madsen L H, Redutoiu S, Frantescu M, Quistgaaid E M H, Miwa H, Downie J A, James E K, Felle H H, Haaning L L, Jensen T H, Sato S, Nakamura Y, Tabata S, Sandal N, Stougaard J. A nucleoporin is required for induction of Ca2+ spiking in legume nodule development and essential for rhizobial and fungal symbiosis. Proc Natl Acad Sci USA,2006,103:359-364
25. Kawaguchi M, Imaizumi-Anraku H, Koiwa H, Niwa S, Ikuta A, Syono K and Akao S. Root, root hair, and symbiotic mutants of the model legume Lotus japonicus. Mol Plant Microbe Interact,2002,15: 17-26
26. Lerouge P, Roche P, Faucher C, et al. Symbiotic host-specificity of Rhizobium meliloti is determined by a sulphated and acylated glucosamine oligosaccharide signal. Nature,1990,344:781-784
27. Limpens E, Mirabella R, Fedorova E, Franken C, Franssen H, Bisseling T and Geurts R. Formation of organelle-like N2-fixing symbiosomes in legume root nodules is controlled by DMI2. PNAS,2005, 102:10375-10380
28. Lodwig E M, Hosie A H, Bourdes A, Findlay K, Allaway D, Karunakaran R, Downie J A, Poole P S. Amino-acid cycling drives nitrogen fixation in the legume-Rhizobium symbiosis. Nature,2003,422: 722-726
29. Madsen E B, Madsen L H, Radutoiu S, Olbryt M, Rakwaiska M, Szczygliwski K, Sato S, Kaneko T, Tabata S, Sandal N and Stougaard J. A receptor kinase gene of the LysM type is involved in legume perception of rhizobial signals. Nature,2003,425:637-640
30. Martinez E, Romero D, Palacios R. The Rhizobium genome. Cri Rev Plant Sci,1990,9:59-93
31. Miwa H, Sun J, Oldroyd G E, Downie J A. Analysis of Nod-Factor-Induced Calcium Signaling in Root Hairs of Symbiotically Defective Mutants of Lotus japonicus. MPMI,2006,19:914-923
32. Moore RC, Cyr RJ. Association between elongation factor-1 alpha and microtubules in vivo is domain dependent and conditional [J].Cell Motil Cytoskeleton,2000,45(4):279
33. Murray JW, Edmonds BT, Liu G, et al. Bundling of actin filaments by elongation factor 1 alpha inhibits polymerization at filament ends [J]. J Cell Biol,1996,135(5):1309
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